1. Field of the Invention
The invention relates to the field of medicine. In particular, it relates to vaccines against flaviviruses and to methods of producing the same.
2. Description of the Related Art
The arthropod-borne flaviviruses are emerging public health problems worldwide. These viruses also pose threats as agents of biowarfare and/or bioterrorism. Japanese encephalitis virus (JEV) is estimated to produce over 50,000 cases of CNS disease a year, about one-third of which result in death (2), making it the flavivirus that causes the highest worldwide mortality.
There is an efficacious inactivated viral vaccine (INV) for Japanese encephalitis virus, but its production has been halted due to adverse events (3). New Japanese encephalitis virus inactivated viral vaccines are in development, and a live-attenuated virus vaccine (LAV) for Japanese encephalitis virus in use in China for decades, has recently been used in other developing countries. There is also a well-characterized rhesus macaque model where JE-INV potency and efficacy correlate with human vaccine potency and efficacy. Large urban epidemics of yellow fever (YF) that plagued tropical and subtropical regions until the early 1900s have been eliminated by mosquito control and application of the efficacious YF-17D live-attenuated virus vaccine.
Although originally thought to be very safe, an alarming number of cases of yellow fever (referred to as acute viscerotropic disease from yellow fever vaccination—YEL-AVD) have been associated with recent yellow fever-17D vaccination campaigns, suggesting that a safer vaccine is needed. Of particular concern are analyses of the viruses recovered from some cases of YEL-AVD (which displayed severe symptoms of hemorrhagic fever indistinguishable from jungle YF) that failed to produce evidence that YEL-AVD has been caused by simple reversion of the vaccine virus (4). Thus, YF-17D vaccine quality control systems similar to those implemented by the WHO in responses to YF-17D-associated neurological disease caused by revertant viruses several decades ago (5,6) are unlikely to be able to make the current YF-17D vaccine safer.
Dengue includes a spectrum of illnesses caused by infection with one of four serotypes of Dengue virus (types 1-4) that occur in many tropical and subtropical regions. The geographic distribution of dengue has expanded over the last 30 years to include more than 100 countries (7). Based on the number of infections with Dengue virus (estimated to be 50 million per year) and the fact that there are 100s-of-thousands of cases of severe dengue each year (7), Dengue virus is considered the most important arthropod-borne virus (7). In some cases, Dengue virus produces a sub-clinical infection, but a febrile illness, dengue fever (DF) occurs in many infected individuals. A portion of dengue fever patients, develop more severe syndromes such as dengue hemorrhagic fever (DHF).
Live-attenuated virus vaccine and inactivated viral vaccine for dengue are in development, but to date, no dengue vaccines have progressed beyond clinical trials, and unique aspects of immunopathogenesis of dengue (8) are problematic for most vaccines in development. No vaccines exist for diseases caused by West Nile virus (WNV), which is responsible for the largest epidemic of viral encephalitis in United States history.
The flavivirus genome is a positive-sense, single-stranded, non-poly(A) RNA of ˜11 kb in length that can initiate infection in the absence of viral proteins. The genome is translated as a single polyprotein that is co- and post-translationally processed by the combined actions of viral and cellular proteases. The 5′ quarter of the genome encodes the structural proteins: capsid (C), membrane (M) (produced from its precursor, prM), and envelope (E). The remaining three quarters of the genome encodes non-structural proteins that replicate the genome, process viral proteins, promote virus assembly, and interact with the host cell. The flavivirus virion is a 50 nm particle consisting of a nucleocapsid surrounded by a lipid bilayer containing E and M. The nucleocapsid, which consists of C and the RNA genome, buds through prM/E-studded patches of the endoplasmic reticulum membrane to produce provirions, which accumulate within the cell's secretory pathway. As these particles transit the secretory pathway of the cell, they mature into viral particles as prM is cleaved to M by a cellular protease (furin). Expression of prM/E in eukaryotic cells results in secretion of 20-30 nm sub-viral particles (SVPs) that share properties with a natural by-product of flavivirus infection (9). Sub-viral particles consist of lipid bilayers containing M and E without C or any genetic material. Sub-viral particles have been demonstrated to be useful subunit vaccines (10), and these particles are considered to be key components produced by many efficacious vaccine candidates (9,11-14).
Flavivirus vaccines in development share problems with existing vaccines. Current inactivated viral vaccine {including the Japanese encephalitis virus inactivated viral vaccine (2.1) and a tick-borne encephalitis (TBE) inactivated viral vaccine in use in Western Europe}, as well as a replacement Japanese encephalitis virus inactivated viral vaccine (15), require extensive purification, and are of low potency, requiring multiple vaccinations. A new flavivirus subunit vaccine candidate (16,17) may suffer from similar problems. Viral-vectored vaccines, including a recently described alphavirus replicon-vectored vaccine (18) may also suffer from problems of “vector immunity” (19) that interfered with the use of vaccinia virus as a vector for a recombinant DNA-derived Japanese encephalitis virus vaccine candidate (20). DNA vaccines have low potency (21). There may also be problems with existing and new live-attenuated virus vaccines including the chimeras generated from YF-17D that relate to an incomplete understanding of the basis of their attenuation, and a resultant concern that they might prove to be unsafe in a portion of the vaccinated population. Specifically, for all live-attenuated virus vaccines in development, there are concerns that like YF-17D (2.1), these vaccines may not be safe in all vaccines, especially the immunocompromised, and in this population (or perhaps even in a small subset of normal vaccines) serious disease will result from these “live” virus vaccines.
Humoral immunity plays a critical role in control of flavivirus infections. Mechanisms of antibody-mediated immunity include: blocking viral binding to cells, Fc receptor-dependent viral clearance, and antibody-mediated cytotoxicity. Although immunity to E, which covers the entire surface of the virion (22), appears to be the primary target of neutralizing (NEUT) antibodies (23), multiple studies have demonstrated that antibodies to NS1 can protect from flavivirus disease (24-26). Consistent with the role of humoral immunity in protection, B cell-deficient mice are more susceptible to flavivirus disease than intact animals (27-30) and protection against flavivirus infection in B cell-deficient mice can be partially restored by adoptive transfer of immune splenocytes (28).
Cellular immunity also plays a role in controlling flavivirus infections. Passive transfer of JEV-immune CTLs has been shown to mediate protection from Japanese encephalitis virus in mice (31), and more recent work with WNV has shown that mice with defects in CTL responses have a reduced ability to clear WNV infections (32,33). In addition, mice that have received WNV-specific CD8+ (34,35) or CD4+ T cells (36) are protected from lethal challenge. Also, CTL responses have been detected in human volunteers given candidate vaccines expressing prM/E/NS1 proteins (37). Although, CTL responses may not be as important as humoral immunity in providing protection from disease, they may be particularly helpful in vulnerable populations, such as the elderly, very young and immunocompromised, where antibody may not be sufficient to confer protection from infectious diseases (38).
The innate immune system is the first line of defense against invading pathogens. One key aspect of this system is recognition of pathogen-associated molecular patterns through pattern-recognition receptors that trigger signaling cascades resulting in secretion of cytokines and chemokines that activate antimicrobial mechanisms and direct adaptive immune responses. Current state-of-the-art vaccinology is trying to systematically characterize and harness this innate/adaptive interface, and it seems likely that advances in this area will translate into better vaccines. The current paradigm reiterates “old-school” thinking that there is likely to be a considerable advantage to the use of self-replicating virus-derived vectors for expression of vaccine antigens (e.g., prM/E and NS1 proteins), since the vector “infections” should induce responses that lead to the type of innate immune stimulation that has evolved to produce an effective adaptive immune response. Therefore, such vectors (e.g., RepliVAX) are likely to provide better protection than inactivated viral vaccines or subunit vaccines by mimicking viral infection and thus stimulating more effective immune responses.
The inventors have recognized a need in the art for a new class of flavivirus vaccines that is superior to other flavivirus vaccine technologies. Specifically, the prior art is deficient in flavivirus vaccine that 1) does not cause disease, even in the immunocompromised, 2) has high potency due to in situ production of immunogens in a way that mimics viral infection, 3) has potential for inexpensive production. The present invention fulfills this long-standing need and desire in the art.